Although over 16 different hypothalamic transcription factors are implicated in controlling fertility, the molecular mechanisms of reproduction remain unclear. The hlh2 basic helix-loop-helix (bHLH) transcription factor is expressed throughout the developing nervous system. In adults, expression is limited to specific neurons in the hypothalamus, pre-optic area and hebenula. Nhlh2 knockout mice (N2KO) are obese and hypogonadal. In addition, they have reduced levels of follicle stimulating hormone and testosterone, and do not show normal male sexual behaviors. N2KO mice also have reduced sperm concentration in their caudal epididymus, but using in vitro fertilization, we have found that sperm from N2KO mice are fully capable of fertilizing oocytes. Thus, infertility in male N2KO mice is largely due to lack of sexual behavior and partially due to reduced sperm production. In other studies, we have found that levels of two neuropeptide processing enzymes, PC1 and PC2, are reduced up to 90% in N2KO mice. These processing enzymes are thought to process GnRH as well as other hypothalamic neuropeptides involved in both fertility and obesity. Based on these preliminary findings, we propose that the Nhlh2 transcription factor regulates male sexual behavior and fertility by up-regulating the expression of PC1and PC2 processing enzymes which are necessary for production of mature GnRH peptide in the pre-optic area of the brain. We will use both animal-based and molecular approaches to test this hypothesis. In the first aim, we will ask if Nhlh2 is co-expressed with GnRH, PC1 and PC2. We will also measure levels of PC1 and PC2 mRNA in pre-optic area neurons, and levels of mature GnRH peptide in N2KO and normal mice. In the second aim, we will supply male N2KO mice with testosterone and ask if this hormone is sufficient to restore male sexual behavior and spermatogenesis. To date, N2KO mice are the only knockout mice in which deletion of a neuronal bHLH transcription factor results in obesity, hypogonadism, and lack of male sexual behavior. Studies of these animals are key to understanding molecular control of male fertility.